Expression of receptors for VEGFs on normal human thyroid follicular cells and their role in follicle formation.

Human thyroid follicular cells in culture expressed the mRNAs for the receptors for vascular endothelial growth factors (VEGFRs). The relative expression was neuropilin1 = neuropilin2 = VEGFR2 > VEGFR1 > VEGFR3. Western blotting for VEGFR2 showed labeling of proteins ~200-230 kDa. Clonal follicular thyroid cell lines (FRTL5 and FTC133) also expressed mRNAs for the VEGFR1 and 2 obviating concerns of endothelial cell contamination. In the primary cultures, TSH, which is essential for expression of differentiated function, reduced VEGFR2 mRNA levels by 60%. Immunostaining for VEGFRs and neuropilin2 (NRP2), showed expression on the plasma membrane but with the exception of neuropilin1 (NRP1), all VEGFRs were also found in the cytoplasm and nucleus. Antibody specific for phosphotyrosine 1214 in VEGFR2 showed that the receptor was phosphorylated in the primary cultures and the cell lines. When VEGFR signaling was blocked with a specific inhibitor, follicle formation in the primary cultures was enhanced suggesting that VEGFR activation was detrimental to follicle formation. Immunostaining of sections of normal thyroids and various pathologies showed staining for VEGFR2 and pVEGFR2. We conclude that normal thyroid follicular cell express VEGFRs. For VEGFR2 its subcellular localization suggests functions additional to that of a cell surface receptor and a role in follicular integrity.

A novel mechanism of sodium iodide symporter repression in differentiated thyroid cancer.

Differentiated thyroid cancers and their metastases frequently exhibit reduced iodide uptake, impacting on the efficacy of radioiodine ablation therapy. PTTG binding factor (PBF) is a proto-oncogene implicated in the pathogenesis of thyroid cancer. We recently reported that PBF inhibits iodide uptake, and have now elucidated a mechanism by which PBF directly modulates sodium iodide symporter (NIS) activity in vitro. In subcellular localisation studies, PBF overexpression resulted in the redistribution of NIS from the plasma membrane into intracellular vesicles, where it colocalised with the tetraspanin CD63. Cell-surface biotinylation assays confirmed a reduction in plasma membrane NIS expression following PBF transfection compared with vector-only treatment. Coimmunoprecipitation and GST-pull-down experiments demonstrated a direct interaction between NIS and PBF, the functional consequence of which was assessed using iodide-uptake studies in rat thyroid FRTL-5 cells. PBF repressed iodide uptake, whereas three deletion mutants, which did not localise within intracellular vesicles, lost the ability to inhibit NIS activity. In summary, we present an entirely novel mechanism by which the proto-oncogene PBF binds NIS and alters its subcellular localisation, thereby regulating its ability to uptake iodide. Given that PBF is overexpressed in thyroid cancer, these findings have profound implications for thyroid cancer ablation using radioiodine.

Oxidative stress and dysregulation of the taurine transporter in high-glucose-exposed human Schwann cells: implications for pathogenesis of diabetic neuropathy.

In human Schwann cells, the role of taurine in regulating glucose-induced changes in antioxidant defense systems has been examined. Treatment with high glucose for 7 days induced reactive oxygen species, increased 4-hydroxynoneal adducts (20 +/- 5%, P < 0.05) and poly(ADP-ribosyl)ated proteins (40 +/- 13%, P < 0.05). Increases in these markers of oxidative stress were reversed by simultaneous incubation in 0.25 mM taurine. Both high glucose and taurine independently increased superoxide dismutase and catalase activity and decreased glutathione levels, but their effects were not additive. Glucose reduced taurine transporter (TauT) mRNA and protein in a dose-dependent manner with maximal decreases of 66 +/- 6 and 63 +/- 12%, respectively (P < 0.05 both). The V(max) for taurine uptake was decreased in 30 mM glucose from 61 +/- 5 to 42 +/- 3 pmol x min(-1) x mg protein(-1) (P < 0.001). Glucose-induced TauT downregulation could be reversed by inhibition of aldose reductase, a pathway that depletes NADPH and increases osmotic stress and protein glycation. TauT protein was increased more than threefold, and the V(max) for taurine uptake doubled (P < 0.05 both) by prooxidants. TauT downregulation was reversed both by treatment with the antioxidant alpha-lipoic acid, which increased TauT mRNA by 60% and V(max) by 50% (P < 0.05 both), and by the aldose reductase inhibitor sorbinil, which increased TauT mRNA 380% and V(max) by 98% (P < 0.01 both). These data highlight the potential therapeutic benefits of taurine supplementation in diabetic complications and provide mechanisms whereby taurine restoration could be achieved in order to prevent or reverse diabetic complications.

Sphingosine 1-phosphate receptor expression profile and regulation of migration in human thyroid cancer cells.

S1P (sphingosine 1-phosphate) receptor expression and the effects of S1P on migration were studied in one papillary (NPA), two follicular (ML-1, WRO) and two anaplastic (FRO, ARO) thyroid cancer cell lines, as well as in human thyroid cells in primary culture. Additionally, the effects of S1P on proliferation, adhesion and calcium signalling were addressed in ML-1 and FRO cells. All cell types expressed multiple S1P receptors. S1P evoked intracellular calcium signalling in primary cultures, ML-1 cells and FRO cells. Neither proliferation nor migration was affected in primary cultures, whereas S1P partly inhibited proliferation in ML-1 and FRO cells. Low nanomolar concentrations of S1P inhibited migration in FRO, WRO and ARO cells, but stimulated ML-1 cell migration. Consistently, S1P1 and S1P3, which mediate migratory responses, were strongly expressed in ML-1 cells, and S1P2, which inhibits migration, was the dominating receptor in the other cell lines. The migratory effect in ML-1 cells was mediated by G(i) and phosphatidylinositol 3-kinase. Both S1P and the S1P1-specific agonist SEW-2871 induced Akt phosphorylation at Ser473. However, SEW-2871 failed to stimulate migration, whereas the S1P1/S1P3 antagonist VPC 23019 inhibited S1P-induced migration. The results suggest that aberrant S1P receptor expression may enhance thyroid cancer cell migration and thus contribute to the metastatic behaviour of some thyroid tumours.

Protein kinase C delta is not activated by caspase-3 and its inhibition is sufficient to induce apoptosis in the colon cancer line, COLO 205.

Activation of protein kinase C delta (PKCdelta) is believed to be pro-apoptotic. PKCdelta is reported to be reduced in colon cancers. Using a colon cancer cell line, COLO 205, we have examined the roles of PKCdelta in apoptosis and of caspase-3 in the activation and inhibition of PKCdelta. PKCdelta activation with bistratene A and its inhibition with rottlerin induced apoptosis. Effects of PKC activators and inhibitors were additive, suggesting that PKCdelta down-regulation was responsible for the effects on apoptosis. Different apoptotic pathways induced PKCdelta cleavage, but the fragment produced was inactive in kinase assays. Caspase-3 inhibition did not block DNA fragmentation or PKCdelta proteolysis despite blocking intracellular caspase-3 activity. Calpain inhibition with calpeptin did not prevent TPA-induced PKCdelta cleavage. We conclude that in colonocytes, inhibition of PKCdelta is sufficient to lead to caspase-3-independent apoptosis. Caspase-3 does not cleave PKCdelta to an active form, nor does caspase-3 inhibition block apoptosis.

Complete inhibition of goiter in mice requires combined gene therapy modification of angiopoietin, vascular endothelial growth factor, and fibroblast growth factor signaling.

In goiter, increased expression of growth factors and their receptors occurs. We have inhibited the action of some of these growth factors, alone and in combination, to determine which are important in goitrogenesis. Recombinant adenovirus vectors (RAds) expressing truncated, secreted forms of human Tie2 (RAd-sTie2) and vascular endothelial growth factor receptor 1 (RAd-sVEGFR1) or a truncated, dominant-negative fibroblast growth factor receptor 1 (RAdDN-FGFR1) were used. Goiters in mice were induced by feeding an iodide-deficient diet, containing methimazole and sodium perchlorate. RAds were administered to mice simultaneously with the goitrogenic regimen, which was continued for 14 d. RAd treatment did not significantly affect increases in TSH or reductions in thyroid hormone or thyroid hyperactivity seen in goitrogen-treated controls mice, suggesting no effect on pituitary or thyroid responses to hypothyroidism. In control goiters, a 4-fold increase in vascular volume accompanied a 2-fold increase in thyroid mass. Complete inhibition of these increases was found when animals were treated with the three RAds in combination. In thyroids from three RAd-treated animals, there was marked, significant inhibition of Tie2, FGFR1, VEGFR1, FGF-2, and VEGF expression, compared with control goiters. When used individually, RAdDN-FGFR1 partially prevented goiter and RAd-sVEGFR1 partially reduced vascular volume. Their effects were not additive. RAd-sTie2 did not reduce goiter mass or vascular volume when used alone but was essential for complete goiter inhibition. VEGF and VEGFR1 expression was reduced in these thyroids. Limitation of physiologic organ growth is complex, requiring inhibition of multiple, interdependent growth factor axes.

Essential role of fibroblast growth factor signaling in preadipoctye differentiation.

We have examined the expression and role of autocrine fibroblast growth factors (FGFs) in human preadipocytes through their differentiation in vitro. A high-molecular weight form of FGF-2 was initially strongly expressed, but 6-9 d after induction of differentiation, its expression decreased markedly. This coincided with the first appearance of visible lipid droplets within the cells. FGF-2 (18 kDa) was not found. FGF receptor (FGFR) 1 was detected as a single band of 125 kDa that also decreased with differentiation. Its decrease preceded that of FGF-2. Despite the decrease in cell-associated FGF-2 with differentiation, secreted FGF-2 was 2.5-fold higher in the differentiated preadipocytes. To determine whether FGF-2 had an autocrine role, FGFR signaling was inhibited using recombinant adenovirus expressing dominant negative FGFR1 (RAdDN-FGFR1) and a specific inhibitor of FGFR1 signaling, PD166866. Preadipocytes transduced with RAdDN-FGFR1 expressed a truncated, 79-kDa FGFR1. Differentiation, assessed by lipid droplet formation, was completely prevented by RAdDN-FGFR1 and by PD166866. The protein content in the cell layer and glucose uptake were significantly reduced by both agents. The insulin-sensitizing drug, rosiglitazone, did not prevent the actions of RAdDN-FGFR1 or PD166866. Controlling adipose tissue growth by limiting FGF actions may provide a means to combat obesity.

Pituitary tumor transforming gene binding factor: a novel transforming gene in thyroid tumorigenesis.

CONTEXT: There are currently no clear markers for the detection of differentiated thyroid cancer and its recurrence. Pituitary tumor transforming gene (PTTG) is a protooncogene implicated in the pathogenesis of multiple tumor types, which stimulates fibroblast growth factor-2 secretion via PTTG binding factor (PBF). OBJECTIVE: The aim of this study was to ascertain whether PBF expression is associated with thyroid cancer outcome. DESIGN: PBF expression was measured at the mRNA and protein level. Tissue was collected during surgery, with normal samples being taken from the contralateral lobe. In vitro studies ascertained the ability of PBF to transform cells and form tumors in nude mice and its subcellular localization. SETTING: The study was conducted at a primary care/referral center. PATIENTS: Thyroid tumors were collected from a series of 27 patients undergoing surgical excision of papillary and follicular thyroid tumors. INTERVENTION: No intervention was conducted. MAIN OUTCOME MEASURE: The expression of PBF in thyroid cancers compared with normal thyroid, hypothesized before the investigation to be raised in tumors, was the main outcome measure. RESULTS: PBF mRNA expression was higher in differentiated thyroid carcinomas than in normal thyroid (P < 0.001; n = 27) and was independently associated with tumor recurrence (P = 0.002; R(2) = 0.49). PTTG was able to up-regulate PBF mRNA expression in vitro (P < 0.001; n = 12), and stable overexpression of PBF in NIH3T3 cells resulted in significant colony formation (P < 0.001; n = 12). In vivo, stable sc overexpression of PBF induced tumor formation in athymic nude mice. CONCLUSIONS: PBF is an additional prognostic indicator in differentiated thyroid cancer that is transforming in vitro and tumorigenic in vivo.

Differential regulation of lipogenesis and leptin production by independent signaling pathways and rosiglitazone during human adipocyte differentiation.

Since leptin levels are independently correlated with risk of coronary heart disease, we have identified signaling pathways important in mediating leptin production and lipogenesis in human preadipocytes. We used inhibitors of p70(S6) kinase, p42/44 mitogen-activated protein kinase (MAPK), p38 MAPK, and phosphatidylinositol 3-kinase (PI3K). Human preadipocytes were induced to differentiate in insulin, dexamethasone, triiodothyronine, and 3-isobutyl-1-methylxanthine in the presence or absence of inhibitors and the peroxisome proliferator-activated receptor (PPAR)-gamma activator rosiglitazone. Differentiation was assessed by measuring leptin secretion, lipid content, and lipogenic activity. Rosiglitazone increased cell protein by 15%, the lipid content of the cell layer was doubled, and the lipogenic activity increased sevenfold but did not stimulate leptin secretion. None of the inhibitors significantly inhibited protein content over 20 days, but lipid content and lipogenic activity were inhibited by p70(S6) kinase and p38 MAPK inhibition but not by p42/44 MAPK or PI3K inhibition. All of the inhibitors significantly decreased leptin secretion, and these inhibitory effects were increased by coincubation with rosiglitazone. We conclude that PI3K and p42/44 MAPK pathways are not critical to the differentiation program leading to lipid accumulation, but stimulation of leptin secretion is dependent on these as well as the p70(S6) kinase and p38 MAPK signaling pathways.

Insulin and rosiglitazone regulation of lipolysis and lipogenesis in human adipose tissue in vitro.

Lipolysis is an important process determining fuel metabolism, and insulin regulates this process in adipose tissue. The aim of this study was to investigate the long-term effects of insulin, an insulin enhancer (rosiglitazone [RSG]), and insulin in combination with RSG on the regulation of lipolysis and lipogenesis in human abdominal subcutaneous fat. Lipolysis and lipogenesis were assessed by protein expression studies of hormone-sensitive lipase (HSL) (84 kDa) and lipoprotein lipase (LPL) (56 kDa), respectively. In addition, lipolytic rate was assessed by glycerol release assay and tumor necrosis factor (TNF)-alpha release measured by enzyme-linked immunosorbent assay (n = 12). In subcutaneous adipocytes, increasing insulin doses stimulated LPL expression, with maximal stimulation at 100 nmol/l insulin (control, 1.0 +/- 0.0 [mean +/- SE, protein expression relative to control]; 1 nmol/l insulin, 0.87 +/- 0.13; 100 nmol/l insulin, 1.68 +/- 0.19; P < 0.001). In contrast, insulin at the 100 nmol/l dose reduced the expression of HSL (100 nmol/l insulin, 0.49 +/- 0.05; P < 0.05), while no significant reduction was observed at other doses. Higher doses of insulin stimulated both HSL (1,000 nmol/l insulin, 1.4 +/- 0.07; P < 0.01) and LPL (control 1.00 +/- 0.0; 1,000 nmol/l insulin, 2.66 +/- 0.27; P < 0.01) protein expression. Cotreatment with RSG induced an increased dose response to insulin for LPL and HSL (P < 0.05); RSG alone also increased LPL and HSL expression (P < 0.05). Insulin stimulated TNF-alpha secretion in a dose-dependent manner (P < 0.01); the addition of RSG (10(-8) mol/l) reduced TNF-alpha secretion (P < 0.05). In summary, chronic treatment of human adipocytes with insulin stimulates lipolysis and LPL protein expression. The addition of RSG reduced the lipolytic rate and TNF-alpha secretion. The increase in lipolysis is not explained by changes in HSL expression. These data, therefore, may explain in part why hyperinsulinemia coexists with increased circulating nonesterified free fatty acids and increased adiposity in obese and/or type 2 diabetic patients.

Adenovirus-mediated expression of dominant negative fibroblast growth factor (FGF) receptor 1 in thyroid cells blocks FGF effects and reduces goitrogenesis in mice.

Levels of fibroblast growth factor 2 (FGF-2) and its receptor, FGFR1, are elevated in goiter, but whether this is a direct effect of TSH is unknown. We have determined the regulation of FGF-2 and FGFR1 synthesis by TSH in a rat thyroid cell line (FRTL5) and have used a replication-defective adenovirus (RAd) expressing dominant negative FGFR1 (RAdDN-FGFR1) to examine the role of FGFR signaling in vitro and in goiter induced in mice. TSH induced FGF-2 and increased the expression of FGFR1 in FRTL5 cells. Infection of TSH-stimulated FRTL5 cells with RAdDN-FGFR1 inhibited growth and prevented FGF-2-mediated inhibition of (125)I uptake. Similar effects were found in primary cultures of human thyroid follicular cells. For in vivo experiments, male BALB/c mice were injected systemically with RAdDN-FGFR1 or RAd encoding green fluorescent protein, and goiter was simultaneously induced. Mouse thyroid follicles were shown to be transduced with RAd encoding green fluorescent protein. Circulating TSH was elevated comparably in the two groups. In the RAdDN-FGFR1-injected animals, goiter induced over 14 d was significantly smaller, and the vascular volume increase seen in goiter was also diminished. We conclude that the FGF axis is important in thyroid growth and that RAdDN-FGFR1 effectively blocks FGF actions, offering a means to control goitrogenesis.

Glucocorticoid regulation of p450 aromatase activity in human adipose tissue: gender and site differences.

The distinct gender-specific patterns of fat distribution in men and women (android and gynoid) suggest a role for sex steroids. In keeping with these observations, it has been suggested that estrogens can promote preadipocyte cell proliferation and/or differentiation. The enzyme aromatase P450 is responsible for the conversion of androgen precursor steroids to estrogens and may, therefore, have a role in regulating adipose tissue mass and its distribution. We have investigated the glucocorticoid regulation of aromatase expression in human adipose tissue, specifically to define any site- and gender-specific differences. Abdominal subcutaneous (Sc) and omental (Om) adipose tissue was obtained from male and female patients undergoing elective surgery. After collagenase digestion, preadipocytes were cultured in serum-free medium, for 6-10 d, until confluent with either cortisol (10(-6) M, 10(-7) M) or insulin (500 nM) or a combination of both treatments. Adipocytes were studied in suspension cultures. Aromatase activity was assessed using tritiated [1 beta-(3)H]-androstenedione as substrate. In Sc preadipocytes, basal aromatase activity increased in females from 11.5 +/- 1.4 (mean plus minus SEM) to 28.0 +/- 1.8 pmol/mg x h (n = 17, P < 0.05) with 10(-6) M cortisol. By contrast, in males, aromatase activity was inhibited by 10(-6) M cortisol (19.4 +/- 2.4 pmol/mg x h vs. 7.5 +/- 1.3, n = 9, P < 0.01; men vs. women, P < 0.005). These data were endorsed through Western blot analysis using an in-house antihuman aromatase antibody, which recognized a specific 55-kDa species. Aromatase activity was less at Om sites in preadipocytes, increasing in females from 1.1 +/- 0.2 to 3.2 +/- 0.7 pmol/mg x h with 10(-6) M cortisol (P < 0.05) and in males from 2.6 +/- 0.1 pmol/mg x h to 7.8 +/- 0.3 pmol/mg x h after cortisol (men vs. women, P < 0.001). Cortisol-induced aromatase activity in Om adipocytes from postmenopausal females was higher than that in premenopausal females (P < 0.001). Insulin had no independent effect on aromatase expression, but coincubation of preadipocytes with cortisol and insulin eliminated both gender- and site-specific differences. In conclusion, in women, but not men, cortisol increased aromatase activity at Sc sites, and this may facilitate predilection for Sc adiposity in females. The observed site-, gender-, and menopausal-specific differences in the glucocorticoid regulation of this enzyme may contribute to the gender- and menopausal-specific patterns of fat distribution.

Targets of 17beta-oestradiol-induced apoptosis in colon cancer cells: a mechanism for the protective effects of hormone replacement therapy?

Epidemiological studies show a strong link between postmenopausal hormone replacement therapy and decreased incidence of colorectal cancer. The colon cancer cell line, COLO 205, develops sensitivity to 17beta-oestradiol (E(2)) in apoptosis assays with increasing passage number (>40), and we hypothesised that genes selectively regulated in multiply passaged cells were likely to be important in E(2)-related apoptosis. Gene array analysis was used to compare the patterns of genes up- or down-regulated in E(2)-sensitive and -insensitive cells. For some genes, changes in mRNA expression were confirmed by protein expression analyses. Changes found in response to E(2) in multiply passaged cells, but not minimally passaged cells, included induction of growth arrest and DNA damage-inducible protein 153 (GADD153), and repression of Kirsten-Ras 2B (K-Ras-2B), metastasis inhibition factor NM23 and vascular endothelial growth factor. A second group of genes was regulated with E(2) exposure in both cell types, and is unlikely to be critically involved in E(2)-associated apoptosis. These included up-regulation of butyrate response factor 1 (BRF1) and down-regulation of c-jun and the breast cancer associated ring domain gene known as BARD1. By comparing control arrays from the two cell populations, cAMP-response element-binding protein (CBP), which is associated with steroid receptor-dependent target gene transcription and the oncoprotein, tyrosine kinase-T3 (TRK-T3), were up-regulated whereas retinoic acid receptor alpha (RARalpha) was down-regulated in multiply passaged cells. This study provides evidence for selective regulation of genes in colon cancer cells by E(2), indicates which of those regulated are likely to be involved in induced apoptosis, and suggests genes likely to be responsible for facilitation.

Local factors regulating growth and function of human thyroid cells in vitro and in vivo.

In vitro culture of thyroid follicles is often employed to study thyroid cell biology and the control of thyroid follicular cell growth. For acceptance as a valid model, cultures should maintain differentiated function, which can be measured as the organification of [Formula: see text] and/or the de novo synthesis of thyroid hormones. In this article, the properties and merits of the various in vitro cultures of thyroid follicular cells and the potential effects of thyroid-specific, secreted products (thyroid hormones, thyroglobulin) and autocrine factors (proteases, growth factors and inhibitors) on thyroid growth and function, are explored. The regulation of the secretion of autocrine/paracrine factors by thyroid follicular cells is reviewed and methods by which cells may defend themselves from the effects of bioactive growth factors are discussed with particular reference to FGF signalling. The role and regulation of plasminogen activator activity, which may be central to the release and/or activation of growth factors and their receptors, and the secretion of angiogenic factors are described.

Taurine reduces nitrosative stress and nitric oxide synthase expression in high glucose-exposed human Schwann cells.

The role of taurine in regulating glucose-induced nitrosative stress has been examined in human Schwann cells, a model for understanding the pathogenesis of diabetic neuropathy. Exposure to high glucose increased nitrated proteins (1.56 fold p<0.05), inducible nitric oxide synthase (iNOS) and neuronal NOS (nNOS) mRNA expression (1.55 fold and 2.2 fold respectively, p<0.05 both), phospho-p38 MAPK (1.32 fold, p<0.05) abundance and decreased Schwann cell growth (11±2%, p<0.05). Taurine supplementation prevented high-glucose induced iNOS and nNOS mRNA upregulation, reduced nitrated proteins and phospho-p38 MAPK (56±11% and 45±18% (p<0.05 both) respectively) and restored Schwann cell growth to control levels. High glucose and taurine treatment alone reduced phospho-p42/44 MAPK and phospho-AKT to below detectable levels. Treatment of human Schwann cells with donors of nitric oxide and peroxynitrite reduced taurine transporter (TauT) expression (by 35±5% and 29±7% respectively p<0.05 both) as well as the maximum velocity of taurine uptake (TauT Vmax). NOS inhibition prevented glucose-mediated TauT mRNA downregulation, and restored TauT Vmax. These data demonstrate an important role for taurine in the prevention of nitrosative stress in human Schwann cells, which may have important implications for the development and treatment of diabetic neuropathy.

Regulation of human thyroid follicular cell function by inhibition of vascular endothelial growth factor receptor signalling.

The potential autocrine role of human thyroid vascular endothelial growth factors (VEGFs) was examined using the VEGF receptor (VEGFR) inhibitor, ZM306416HCl. ZM306416HCl reduced VEGFR2 phosphorylation and inhibited endogenous, steady-state levels of p42/44 MAPK phosphorylation. It potently inhibited the secretion of plasminogen activators (PA) and increased (125)I uptake. Cell survival was compromised but rescued with insulin and TSH. Although the EGF receptor remained responsive to challenge by EGF in p42/44 MAPK assays, stimulatory effects of EGF on PA production were prevented by ZM306416HCl and those of protein kinase C stimulator, TPA reduced. In assays of (125)I uptake, ZM306416HCl prevented the inhibitory effects of EGF but not those of TPA. We conclude that autocrine VEGF may modulate thyroid function and that VEGFR inhibition increases iodide uptake and decreases PA production through regulation of p42/44 MAPK phosphorylation. VEGFR inhibition may have effects on thyroid function which may contribute to "off target" effects in clinical trials.

Proto-oncogene PBF/PTTG1IP regulates thyroid cell growth and represses radioiodide treatment.

Pituitary tumor transforming gene (PTTG)-binding factor (PBF or PTTG1IP) is a little characterized proto-oncogene that has been implicated in the etiology of breast and thyroid tumors. In this study, we created a murine transgenic model to target PBF expression to the thyroid gland (PBF-Tg mice) and found that these mice exhibited normal thyroid function, but a striking enlargement of the thyroid gland associated with hyperplastic and macrofollicular lesions. Expression of the sodium iodide symporter (NIS), a gene essential to the radioiodine ablation of thyroid hyperplasia, neoplasia, and metastasis, was also potently inhibited in PBF-Tg mice. Critically, iodide uptake was repressed in primary thyroid cultures from PBF-Tg mice, which could be rescued by PBF depletion. PBF-Tg thyroids exhibited upregulation of Akt and the TSH receptor (TSHR), each known regulators of thyrocyte proliferation, along with upregulation of the downstream proliferative marker cyclin D1. We extended and confirmed findings from the mouse model by examining PBF expression in human multinodular goiters (MNG), a hyperproliferative thyroid disorder, where PBF and TSHR was strongly upregulated relative to normal thyroid tissue. Furthermore, we showed that depleting PBF in human primary thyrocytes was sufficient to increase radioiodine uptake. Together, our findings indicate that overexpression of PBF causes thyroid cell proliferation, macrofollicular lesions, and hyperplasia, as well as repression of the critical therapeutic route for radioiodide uptake.

Molecular regulation of thyroid gland function.

PURPOSE OF REVIEW: For de-novo thyroid hormone synthesis ex vivo, thyroid follicular cells require a serum-free medium supplying nutrients, iodide, thyroid-stimulating hormone and insulin-like growth factor I (IGF-I) (or insulin). Under these conditions, T3 and T4 are secreted but so are other factors such as growth factors, plasminogen activators, their inhibitors known as serpins, and so on. What is the function of these factors? Do thyroid cells respond to them or are these paracrine/endocrine factors? The purpose of this review is to highlight the current developments in the identification and role of the signalling pathways that regulate thyroid growth and function and the putative role of endogenous thyroid proteases in regulating this. RECENT FINDINGS: The roles of the mitogen-activated protein kinases and phosphoinositol 3 kinases and integrins in mediating growth and function in thyroid cancer cells and the roles of plasminogen activators, their receptors and the downstream signalling pathways they modulate have been developed. Discoveries of novel proteases, expressed in thyroid cancers, may be useful in diagnosis. SUMMARY: The signalling pathways regulating thyroid activity are examined and the roles of follicular cell products in maintaining thyroid homeostasis evaluated. The possibility that thyroid cell products other than T3 and T4 may circulate and have extrathyroidal effects is proposed.

Pituitary tumor transforming gene binding factor: a new gene in breast cancer.

Pituitary tumor transforming gene (PTTG) binding factor (PBF; PTTG1IP) is a relatively uncharacterized oncoprotein whose function remains obscure. Because of the presence of putative estrogen response elements (ERE) in its promoter, we assessed PBF regulation by estrogen. PBF mRNA and protein expression were induced by both diethylstilbestrol and 17beta-estradiol in estrogen receptor alpha (ERalpha)-positive MCF-7 cells. Detailed analysis of the PBF promoter showed that the region -399 to -291 relative to the translational start site contains variable repeats of an 18-bp sequence housing a putative ERE half-site (gcccctcGGTCAcgcctc). Sequencing the PBF promoter from 122 normal subjects revealed that subjects may be homozygous or heterozygous for between 1 and 6 repeats of the ERE. Chromatin immunoprecipitation and oligonucleotide pull-down assays revealed ERalpha binding to the PBF promoter. PBF expression was low or absent in normal breast tissue but was highly expressed in breast cancers. Subjects with greater numbers of ERE repeats showed higher PBF mRNA expression, and PBF protein expression positively correlated with ERalpha status. Cell invasion assays revealed that PBF induces invasion through Matrigel, an action that could be abrogated both by siRNA treatment and specific mutation. Furthermore, PBF is a secreted protein, and loss of secretion prevents PBF inducing cell invasion. Given that PBF is a potent transforming gene, we propose that estrogen treatment in postmenopausal women may upregulate PBF expression, leading to PBF secretion and increased cell invasion. Furthermore, the number of ERE half-sites in the PBF promoter may significantly alter the response to estrogen treatment in individual subjects.

Regulation of plasminogen activators in human thyroid follicular cells and their relationship to differentiated function.

Human thyroid cells in culture take up and organify (125)I when cultured in TSH (acting through cAMP) and insulin. They also secrete urokinase (uPA) and tissue-type (tPA) plasminogen activators (5-100 IU/10(6)cells/day). TSH and insulin both decreased secreted PA activity (PAA), uPA and tPA protein and their mRNAs. Autocrine fibroblast growth factor increased secreted PAA and inhibited thyroid cell (125)I uptake. Epidermal growth factor (EGF) and the protein kinase C (PKC) activator, TPA significantly increased PAA and inhibited thyroid differentiated function, (TPA > EGF). For TPA, effects were rapid, increased PAA secretion and decreased (125)I uptake being seen at 4 h whereas for EGF, a 24 h incubation was required. qRT-PCR showed significantly increased mRNA expression of uPA with lesser effects on tPA. Aprotinin, which inhibits PAA, increased (125)I uptake but did not abrogate the effects of TPA and EGF. The MEKK inhibitor, PD98059 partially reversed the effects of EGF and TPA on PAA, and largely reversed the effects of EGF but not TPA on differentiated function. PKC inhibitors bisindoylmaleimide 1, and the specific PKCbeta inhibitor, LY379196 completely reversed the effects of TPA on (125)I uptake and PAA whereas EGF effects were unaffected. TPA inhibited follicle formation and this effect was blocked by LY379196 but not PD98059. We conclude that in thyroid cells, MAPK activation inversely correlates with (125)I uptake and directly correlates with PA expression, in contrast to the effects of cAMP. TPA effects on iodide metabolism, dissolution of follicles and uPA synthesis are mediated predominantly through PKCbeta whereas EGF exerts its effects through MAPK but not PKCbeta.